This invention relates to an improved method of in vitro fertilisation (hereinafter designated IVF).
Since the first IVF pregnancy was delivered in 1978, this procedure has resulted in thousands of pregnancies and opened a vast new frontier of research and treatment for the infertile couples. Still, there is a significant need for improved infertility treatment modalities today. It is presumed that about one out of seven couples experience problems with subfertility or infertility.
IVF of human oocytes has become commonly used for the treatment of female and male subfertility. The standard IVF treatment includes a long phase of hormone stimulation of the female patient, e.g. 30 days, which is initiated by suppressing the patient""s own follicle stimulating hormone (hereinafter designated FSH) and luteinising hormone (hereinafter designated LH) by gonadotropin releasing hormone (hereinafter designated GnRH), and this is followed by injections of exogenous gonadotropins, e.g. FSH and/or LH, in order to ensure development of multiple preovulatory follicles and aspiration of multiple in vivo matured oocytes immediately before ovulation. The aspirated oocyte is subsequently fertilised in vitro and cultured, typically for three days before transferral back into the uterus at the 4-8 cell stage. Continuous efforts have been made to optimise and simplify this procedure. Nevertheless, the overall pregnancy rate cannot be increased significantly over about 20% with the current treatment modalities. In a large European survey of IVF patients, it was found that 7.2 oocytes out of 11.5 aspirated oocytes per patient had undergone resumption of meiosis immediately before fertilisation, only 4.3 oocytes were fertilised and only 2.2 oocytes reached the 8-cell embryo stage after fertilisation and in vitro culture (ESHRE, Edinburgh, 1997).
Due to the very unpredictable quality of the state of the art embryos today, more than one embryo has to be transferred just to give a reasonable chance of success. Therefore, it is common to transfer 2-3 embryos (up to 5 embryos in some countries), which carries the very large side effect of multiple pregnancies with great discomfort and risk to both patient and children. Moreover, it has been estimated that the increased health care expenses due to multiple birth (twins, triplets etc.) is exceeding the entire IVF expenses.
Hence, there are several disadvantages with the current treatment, the four most notable being:
1. the risk of ovarian hyperstimulation with injecting gonadotropins which is a potential fatal condition that requires hospitalisation,
2. multiple pregnancies (50-1.000 times the normal frequency of twins and triplets, respectively),
3. the existence of considerable patient segments that do not tolerate the current method due to, e.g. polycystic ovarian syndrome and many diabetics,
4. a potential long-term cancer risk.
Furthermore, weight gain, bloating, nausea, vomiting, labile mood and other patient discomforts together with patient reluctance to inject themselves are reported as disadvantages.
It is known from WO 96/00235, corresponding to U.S. Pat. No. 5,716,777, that certain sterol derivatives can be used for regulating meiosis. An examples of such a sterol is 4,4-dimethyl-5xcex1-cholesta-8,14,24-triene-3xcex2-ol (hereinafter designated FF-MAS).
Herein, the term MAS compounds designates compounds which mediate the meiosis of oocytes. More specifically, MAS compounds are compounds which in the test described in Example 1 below has a percentage germinal vesicle breakdown (hereinafter designated GVB) which is significantly higher than the control. Preferred MAS compounds are such having a percentage GVB of at least 50%, preferably at least 80%.
Examples of MAS compounds are mentioned in WO 96/00235, corresponding to U.S. Pat. No. 5,716,777, which describes, inter alia, sterol derivatives, WO 96/27658, corresponding to U.S. Pat. No. 5,830,757, which describes, inter alia, inhibitors of enzymes involved in the biosynthesis of cholesterol, for example, lanosterol 1.4 reductase, 4-demethylase, and lanosterol 8-7-isomerase, and endogenous meiosis activating substances, for example, FF-MAS and 4xcex2-methylzymosterol, WO 97/00884, corresponding to U.S. patent application Ser. No. 08/973,661, which describes, inter alia, sterol derivatives, WO 98/28323, corresponding to U.S. patent application Ser. No. 09/333,391, which describes, inter alia, sterol derivatives, WO 98/52965, corresponding to U.S. Pat. No. 6,177,240, which describes, inter alia, 20-aralkyl-5xcex1-pregnane derivatives, and WO 98/55498, corresponding to U.S. Pat. No. 6,262,282, which describes, inter alia, 17xcex2-allyloxy(thio)alkyl-andostrane derivatives, more specifically in claim 1 thereof.
In WO 95/000265, some potential meiosis regulating substances were tested on immature female mice. 48 hours before the test animal were killed by cervical dislocation, they were given a single injection of human menopausal gonadotropin containing 20 IU FSH and 20 IU LH. The ovaries were removed, placed in a hypoxanthine medium and freed of extraneous tissue. Then, the oocytes were punctured out of the follicles, freed from cumulus cells and cultured in a medium containing a meiosis regulating derivative.
At present, in vitro maturation in humans has proven highly unsuccessful despite substantial interest and clinical efforts.
One object of the present invention is to treat human infertility.
Another object of the present invention is to improve the maturation of human oocytes.
Another object of the present invention is to improve the synchrony of nuclear, cytoplasmic and/or membranous oocyte maturation.
Another object of the present invention is to improve the fertility of oocytes.
Another object of the present invention is to improve the rate of implantation of oocytes by human in vitro maturation and fertilisation.
Another object of the present invention is to diminish the incidence of human preembryos with chromosome abnormalities (aneuploidy).
Another object of the present invention is to improve the cleavage rate of human preembryos.
Another object of the present invention is to improve the quality of human preembryos.
It has now, surprisingly, been found that the IVF can be improved substantially when a MAS compound is added at the stage in the usual method of performing in vitro fertilisation where one would expect that the maturation had taken place in vivo.
Briefly, the present invention relates to a method for human in vitro fertilisation wherein a woman, within a consecutive period of 30 days, is treated with a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative thereof where after oocytes are aspirated and actively final matured or the oocyte maturation is synchronised in vitro in contact with a MAS compound. Preferred embodiments of this invention are those stated in the sub claims below.
Referring to the female cycle, on way of performing the treatment of this invention is as follows:
Around day 21 in one cycle to around day 15 in the following cycle: The eggs are stimulated by treating the woman with GnRH, e.g. Synarel (400-600 xcexcg per day).
Around days 6-15 in the second cycle: The eggs are stimulated by treating the woman with FSH, e.g. Gonal-F, Puregon or Humegon (150-400 IU per day).
Around days 15-16 in the second cycle: The eggs are stimulated by treating the woman with hCG, e.g. Pregnyl or Profasi (2000-5000 IU per day).
Around day 18 in the second cycle: The eggs are retrieved from the woman.
Around day 18-19 in the second cycle: The eggs are maturated with a MAS compound in order to stimulate the meiosis. In this additional maturation step, the concentration of MAS compound may be in the range about 0.1-100 xcexcmol per liter, e.g. 10-20 xcexcmol per liter. The time for this maturation step may be in the range around 1-60 hours. If the preovulatory follicles are induced to luthenise with a lutenising hormone or an agonist or antagonist thereof or an active derivative thereof and/or human chorion gonadotropins or an agonist or antagonist thereof or an active derivative thereof, the maturation of the oocytes with the MAS compound is for a duration of about 1-15 hours, preferably about 6 hours. If, however, preovulatory follicles are not induced to lutenise with a lutenising hormone or an agonist or antagonist thereof or an active derivative thereof and/or human chorion gonadotropins or an agonist or antagonist thereof or an active derivative thereof, the maturation of the oocytes with the MAS compound is for duration of about 15-60 hours.
Around days 19-21 in the second cycle: The eggs are fertilised in vitro.
From the day before aspiration, the woman will receive an oestrogen, e.g., oestrogen valerate (2xc3x9710 mg daily). Two days later, she will also receive a progestogen, e.g., Progestane vagetoria, daily, which will render the lining of the uterus more prone to receive the future embryos. The duration of this treatment will be individually designed per patient. The doctor can chose among a variety of oestrogens and progestogens.
Around day 21 in the second cycle: One or more embryos are transferred to the woman""s uterus.
The description above is designated MAS add-on to the existing IVF protocol to improve efficacy by mediating a final or complete maturation or synchrony in the oocyte. Alternatively, MAS can be used to rescue oocytes in cycles that otherwise would be cancelled due to apparent FSH hyper response. In this instance, the responsible clinician would consider cancellation based on the estradiol profile, ultra-sonography (PCO like response) thus avoiding the hCG treatment. The oocytes are aspirated at the time around day 15-16 substituting hCG treatment.
Apart from the additional maturation step with a MAS compound, the above IVF is performed the usual way. Since one expects that by the traditional IVF procedure the eggs had been matured sufficiently, one would not expect that it would have any additional effect to add this additional maturation step.
Most of the steps in the above treatment and procedure are performed in a known manner and the remaining steps are performed in a manner known per se. More details about the removal of the oocytes from follicles in the ovary, culturing of the isolated oocytes, the culture medium to be used, the fertilisation with sperm, and the transfer of the embryo to the fallopian tube can be found in the literature, for example, in U.S. Pat. No. 5,693,534 which is hereby incorporated by reference.
According to this invention, the MAS compound is added to the culture medium used. In this medium, the amount of the MAS compound is in the range from about 0.01 to about 100 xcexcM, preferably in the range from about 0.1 to about 100 xcexcM.
A preferred reason for treating a woman, within a consecutive period of 30 days, with a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative is to obtain multiple preovulatory follicles.
Hypothalamic hormones are hormones present in the human hypothalamus. Pituitary hormones are hormones present in the human pituitary gland. Gonadotropic hormones are hormones secreted by the anterior lobe of the pituitary in vertebras and by mammalian placenta, which control the activity of gonads. Chemically, they are glycoproteins. Examples of gonadotropic hormones are FSH, LH and chorion gonadotropin, e.g. human chorion gonadotropin (hereinafter designated hCG). FSH stimulates growth of ovarian follicles and their oocytes in ovary and the formation of spermatozoa in testis. FSH can, e.g., be menopausal FSH or recombinant FSH. In females, LH activates the oestrogen-producing tissue of the ovaries to produce progesterone, probably promotes the final stages of the development of ovarian follicles, initiates the final oocyte maturation, induces ovulation and in mammals initiates corpus luteum development. These hormones are known. It is obvious for the skilled art worker that, alternatively, agonists or antagonists of these hormones can be used. It is also obvious for the skilled art worker that, alternatively, active analogues of these hormones can be used. Some of these agonists, antagonists and analogues are known and other can be prepared by process known per se. Examples of such known processes are chemical synthesis and genetic engineering.
In a preferred embodiment, the present invention relates to a method or use wherein the consecutive period of 30 days within which the woman is treated with a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative thereof is at least about 7 days, preferably at least about 10 days, more preferred at least about 14 days.
In another preferred embodiment, the present invention relates to a method or use wherein the woman is treated for infertility, and/or for improving the maturation of her oocytes, and/or for improving the synchrony of nuclear, cytoplasmic and/or membranous oocyte maturation, and/or for improving the fertility of her oocytes, and/or for improving the rate of implantation by human in vitro maturation and fertilisation
In another preferred embodiment, the present invention relates to a method or use wherein the consecutive period is one menstrual cycle.
In another preferred embodiment, the present invention relates to a method or use wherein the maturation of the oocytes with the MAS compound is for a duration of about 15 to about 60 hours.
In another preferred embodiment, the present invention relates to a method or use wherein preovulatory follicles are induced to lutenise with a luteinising hormone (LH) or an agonist or antagonist thereof or an active derivative thereof and/or human chorion gonadotropin (HCG) or an agonist or antagonist thereof or an active derivative thereof.
In another preferred embodiment, the present invention relates to a method or use wherein the maturation of the oocytes with the MAS compound is for a duration of about 1 to about 15 hours, preferably about 6 hours.
In another preferred embodiment, the present invention relates to a method or use wherein the dosage of MAS compound used is about 0.01 to about 100 xcexcmol per liter, preferably about 0.1 to about 100 xcexcmol per liter.
In another preferred embodiment, the present invention relates to a method or use wherein the MAS compound is one of the compounds mentioned in WO 96/00235 (corresponding to U.S. Pat. No. 5,716,777), WO 96/27658 (corresponding to U.S. Pat. No. 5,830,757), WO 97/00884 (corresponding to U.S. patent application Ser. No. 08/973,661), WO 98/28323 (corresponding to U.S. patent application Ser. No. 09/333,391), WO 98/52965 (corresponding to U.S. Pat. No. 6,177,240) and WO 98/55498 (corresponding to U.S. Pat. No. 6,262,282), more specifically compounds mentioned in claim 1 thereof.
In another preferred embodiment, the present invention relates to a method or use wherein the MAS compound is FF-MAS.
Additionally, the present invention relates to the use of a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative thereof in the manufacture of a hormone product which is to be administered to a woman who, within a consecutive period of 30 days, is treated with a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative thereof, and from whom, immediately after said period, one or more oocytes are aspirated, where after said oocyte(s) is/are cultivated in a convenient medium containing a MAS compound as defined herein, where after said oocyte(s) is/are fertilised with human sperm, and where after the resulting embryo(s) is/are transferred to a woman.
Additionally, the present invention relates to the use of a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative thereof and of a MAS compound for the manufacture of a medicament for the treatment of human in vitro fertilisation wherein a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative thereof is, within a consecutive period of 30 days, used to treat a women and, thereafter, the MAS compound is used in an in vitro oocyte maturation of the egg or eggs retrieved from this woman.
Additionally, the present invention relates to a pharmaceutically kit in unit dosage form for use by in vitro fertilisation comprising separate unit dosages, said kit comprising separate dosage units for sequential daily administration of a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative thereof for sequential daily administration and 1 dosage units of a MAS compound. This kit may have the preferred features described above.
The present invention is further illustrated by the following examples, which, however, are not to be construed as limiting. The features disclosed in the foregoing description, in the following examples and in the claims may, both separately and in any combination thereof be material for realising the invention in diverse forms thereof.